Field of Invention
The present invention relates to probiotic food additives for livestock, particularly ruminants. Selected strains of a currently unspeciated bacterium of the genus Paenibacillus are demonstrated to have increased nitrate- and nitrite-reducing capability compared to wild-type strains and other nitrate-reducing bacteria. Ruminants that have consumed toxic amounts of nitrate, which is converted to nitrite during the digestive process, can suffer from nitrate/nitrite toxicity. Thus, the selected strains can be utilized to reduce nitrate/nitrite toxicity occurring in such animals and be used prophylactically to prevent nitrate/nitrite toxicity in animals consuming high-nitrate diets. As high-nitrate diets have been demonstrated to reduce methane production in the rumen, selected strains of the bacterial species described herein can be used in conjunction with such diets to prevent nitrate/nitrite toxicity while reducing methanogenesis. Furthermore, the bacterial strains can cause reductions in unwanted bacteria and food-borne pathogens, such as Escherichia coli and Campylobacter jejuni. 
Background
Intoxication by nitrates present in forages has been recognized in ruminants for a long time. Nitrates are ubiquitous in feed and water and are essentially nontoxic. However, when nitrates are reduced to nitrites in the rumen, nitrites can be present in deadly concentrations. Nitrates become toxic to ruminants when biologically reduced to nitrite by rumen microorganisms that use nitrate as an anaerobic electron acceptor, thus, nitrites can be present in deadly concentrations in food and water. As nitrate reduction increases, the rate of nitrate reduction to nitrite exceeds the rate of nitrite reduction to ammonia, resulting in accumulation of toxic levels of nitrite. The nitrite produced is rapidly absorbed into the bloodstream where it converts hemoglobin to methemoglobin, resulting in loss of oxygen transporting capacity of the blood, anoxia, and can ultimately result in death by asphyxiation. Currently, nitrate poisons countless cattle every year, creating waste and economic burden on livestock producers.
Several interventions are available to control nitrate/nitrite poisoning, but these are generally limited to inconvenient and labor-intensive animal and plant management strategies. For example, reducing nitrogen fertilization, timing harvest based on precipitation, and use of specific harvesting techniques can reduce the amount of nitrate, but cannot eliminate already-accumulated nitrates. However, silage grown under stressful conditions such as drought conditions can lead to an increased levels of nitrate. Due to the risk of nitrate poisoning, a significant amount of forage is deemed unacceptable for consumption. Hundreds of millions of tons of silage across the United States accumulated high nitrate content during droughts occurring between 2011 and 2012. This led to millions of dollars of loss to the beef and dairy industry. Additionally, it is theorized that the conditions caused by global climate change will be precipitous for high nitrate levels in forage in the future. It is likely, therefore, that the loss of forage and livestock will increase as a result. Thus, the present invention seeks to address this issue by decreasing the potential for nitrate poisoning in ruminants through the use of probiotic nitrate- and nitrite-reducing bacterial strains. The present invention also seeks to address the issue by enabling the consumption of low-quality, high-nitrate feed for efficient usage of available resources.
Avoiding nitrate/nitrite poisoning can also be achieved via animal management strategies. Animal management strategies include feeding practices (e.g., diluting high nitrate feeds with low nitrate feeds, gradually increasing from low to high nitrate feed, and supplementing diets of high nitrate feed with grain). However, young or nutritionally stressed animals may not benefit from such practices. Additionally, these techniques are costly and can result in reduced animal performance, as well as forage health and quantity.
Ruminants that consume a high nitrate diet have been reported to adapt and reduce nitrate to nitrite, and reduce nitrite to ammonia, more rapidly due to selection and induction of microbial nitrate reduction activity. Thus, adding specific nitrate- and nitrite-reducing microorganisms to the diet of livestock, with the intent of altering the nitrite reducing capacity of the gastrointestinal tract microbiome, has long been in practice. Although the mode of action of these direct-fed microbial products and their beneficial effect has not always been scientifically demonstrated, the important role microorganisms play in fermentation and digestion is well recognized. The use of nitrate-respiring Propionibacterium species has been described as a direct-fed microbial agent to prevent ruminal nitrate intoxication (see, e.g., U.S. Pat. No. 6,120,810), however, this approach has not gained widespread use due to limitations in the technology. Thus, one aspect of the present invention is to address these technological limitations and provide a bacterial probiotic that can be readily packaged and delivered to livestock, including ruminants.
Methanogenesis is the main route of hydrogen (H2) disposal during the process of rumen fermentation (Beauchemin et al., Aus. J. Exper. Agriculture, (2008) 48:21-27). Removal of H2 from the rumen environment is essential for the efficient continuation of rumen fermentation, but the methane resulting from methanogenesis has been implicated both as a loss of dietary energy to the animal (Johnson and Johnson, J. Anim. Sci., (1995) 73:2483-92) as well as a significant greenhouse gas contributing to global warming (Steinfeld et al., Food and Agriculture Org. of the UN, “Livestock's Long Shadow: Environmental Issues and Options, (2006)). Thus, a solution to both of these problems—efficient conversion of livestock feed to animal production and reducing greenhouse gas emission—would prove both economically and ecologically beneficial.
The production of methane within the digestive tract of ruminants is a sizeable inefficiency. Economically, this energy loss costs the U.S. cattle industry nearly one million dollars a day. Additionally, it is estimated that approximately twenty percent of the powerful greenhouse gas produced each year originates from enteric fermentation, with commercial ruminants being the major source. It is, therefore, an object of the present invention to address the need to reduce methanogenesis in livestock, including ruminants.
The addition of nitrate to the diet of livestock has been demonstrated to reduce methanogenesis (van Zijderveld et al., J. Dairy Sci., (2011) 94:4028-38; Guo et al., J. Anim. Sci., (2009) 22:542-49; Sar et al., J. Anim. Sci., (2005) 83:644-52; Takahashi et al., Anim. Feed Sci. Technol., (1998) 74:273-80; U.S. Pub. No. 2012/0219527; WO 2012/159186). However, the use of nitrate as an alternative hydrogen sink to reduce methanogenesis in the rumen is problematic given that high levels of nitrate in livestock feed can result in nitrate/nitrite poisoning and animal death. Additionally, nitrite accumulation in the rumen can lead to reduced microbial activity in that environment, which leads to reduced feed intake and slower animal growth. Multiple approaches have been taken to alleviate the inhibition of fermentation by nitrite, while also using nitrate to reduce methanogenesis.
Supplementing feed with formate, lactate or fumarate has been suggested (Asanuma et al., J. Dairy Sci., (1999) 82:780-87; Iwamoto et al., Anim. Sci. J., (2001) 72:117-25). Additionally, the co-administration of nitrates with β1-4 galacto-oligosaccharides or nisin (a polycyclic antibacterial polypeptide), has been reported to maintain safe levels of nitrite and methemoglobin while maintaining the beneficial effects of nitrate on methanogenesis (Sar et al., Anim. Feed Sci. Tech., (2004) 115:129-42). However, these chemical additives are not cost effective as they require constant reapplication. Therefore, the strains and methods presented herein provide a mechanism to decrease methanogenesis in livestock using high-nitrate diets, while concomitantly avoiding nitrate/nitrite poisoning in an economical manner.
Enhancement of nitrite reduction in ruminal environments utilizing probiotics has been demonstrated both in vitro and in vivo (Sar et al., (2005) supra; U.S. Pat. No. 6,120,810). A range of bacterial species and combinations of strains has been suggested, including E. coli, Propionibacterium acidiproprionici, coryneform bacteria, Bacillus subtilis, Methylophilus, Actinomyces, ruminal bacteria and various combinations. However, these bacteria have not come into common usage for several reasons. First, certain species such as E. coli are potential human pathogens and are known to cause food-borne illnesses. Thus, the use of such probiotic species is untenable. Second, certain species, such as P. acidiproprionici, may actually increase the conversion of nitrate to nitrite faster than normal, exacerbating the toxicity of nitrate-enhanced feeds. Such strains may also be difficult to incorporate into feeds or feed supplements. Thus, one objective of the present invention is to provide a stable probiotic that is economically feasible, non-pathogenic, and effective at preventing or reducing nitrate/nitrite toxicity.